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Cyclic AMP (cAMP) is a ubiquitous second messenger synthesized by most living organisms. In bacteria, it plays highly diverse roles in metabolism, host colonization, motility, and many other processes important for optimal fitness. The main route of cAMP perception is through transcription factors from the diverse and versatile CRP–FNR protein superfamily. Since the discovery of the very first CRP protein CAP in Escherichia coli more than four decades ago, its homologs have been characterized in both closely related and distant bacterial species. The cAMP-mediated gene activation for carbon catabolism by a CRP protein in the absence of glucose seems to be restricted to E. coli and its close relatives. In other phyla, the regulatory targets are more diverse. In addition to cAMP, cGMP has recently been identified as a ligand of certain CRP proteins. In a CRP dimer, each of the two cyclic nucleotide molecules makes contacts with both protein subunits and effectuates a conformational change that favors DNA binding. Here, we summarize the current knowledge on structural and physiological aspects of E. coli CAP compared with other cAMP- and cGMP-activated transcription factors, and point to emerging trends in metabolic regulation related to lysine modification and membrane association of CRP proteins.

In bacteria, Crp-Fnr superfamily transcription factors are the most ubiquitous receptor proteins of 3’,5’-cyclic adenosine monophosphate (cAMP) and 3’,5’-cyclic guanosine monophosphate (cGMP). The prototypicEscherichia coliCAP protein represents the main CRP subclass and is known to bind cAMP and cGMP, but to mediate transcription activation only in its cAMP-bound state. In contrast, both cyclic nucleotides mediate transcription activation by CRP subclass G protein Clr ofSinorhizobium meliloti. We present crystal structures of apo-Clr, and Clr•cAMP and Clr•cGMP bound to the core motif of the palindromic Clr DNA binding site (CBS). We show that both cyclic nucleotides shift ternary Clr•cNMP•CBS-DNA complexes to almost identical active conformations. Unlike the situation known for theE. coliCAP•cNMP complex, in the Clr•cNMP complex, the nucleobases of cGMP and cAMP are in thesyn-andanti-conformation, respectively, allowing a shift to the active conformations in both cases. Isothermal titration calorimetry measured similar affinities of cAMP and cGMP binding to Clr in presence of CBS core motif DNA (KDcNMP16 μM). However, different affinities were determined in absence of this DNA (KDcGMP24 μM; KDcGMP6 μM). Sequencing of Clr co-immunoprecipitated DNA as well as Electrophoretic Mobility Shift and promoter-probe assays expanded the list of experimentally proven Clr-regulated promoters and CBS. This comprehensive set of CBS features conserved nucleobases, which are in agreement with the sequence readout through interactions of Clr amino acid residues with these nucleobases, as revealed by the Clr•cNMP•CBS-DNA crystal structures.IMPORTANCECyclic 3’,5’-adenosine monophosphate (cAMP) and cyclic 3’,5’-guanosine monophosphate (cGMP) are both long known as important nucleotide second messenger in eukaryotes. This is also the case for cAMP in prokaryotes, whereas a signaling role for cGMP in this domain of life has been recognized only recently. Catabolite repressor proteins (CRPs) are the most ubiquitous bacterial cAMP receptor proteins.Escherichia coliCAP, the prototypic transcription regulator of the main CRP subclass, binds both cyclic mononucleotides, but only the CAP•cAMP complex promotes transcription activation. In contrast, CRP subclass G proteins studied so far, are activated by cGMP, or both by cAMP and cGMP. Here, we report a structural analysis of the bifunctional cAMP- and cGMP-activatable Clr fromSinorhizobium meliloti, how binding of cAMP and cGMP shifts Clr to its active conformation, and the structural basis of its DNA binding site specificity.

When subjected to nutritional stress, bacteria modify their amino acid metabolism and cell division activities by means of the stringent response, which is controlled by the Rsh protein in alphaproteobacteria. An important group of alphaproteobacteria are the rhizobia, which fix atmospheric N2 in symbiosis with legume plants. Although nutritional stress is common for rhizobia while infecting legume roots, the stringent response has scarcely been studied in this group of soil bacteria. In this report, we obtained a mutant with a kanamycin resistance insertion in the rsh gene of Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean. This mutant was defective for type 3 secretion system induction, plant defense suppression at early root infection, and nodulation competition. Furthermore, the mutant produced smaller nodules, although with normal morphology, which led to lower plant biomass production. Soybean (Glycine max) genes GmRIC1 and GmRIC2, involved in autoregulation of nodulation, were upregulated in plants inoculated with the mutant under the N-free condition. In addition, when plants were inoculated in the presence of 10 mM NH4NO3, the mutant produced nodules containing bacteroids, and GmRIC1 and GmRIC2 were downregulated. The rsh mutant released more auxin to the culture supernatant than the wild type, which might in part explain its symbiotic behavior in the presence of combined N. These results indicate that the B. diazoefficiens stringent response integrates into the plant defense suppression and regulation of nodulation circuits in soybean, perhaps mediated by the type 3 secretion system. IMPORTANCE The symbiotic N2 fixation carried out between prokaryotic rhizobia and legume plants performs a substantial contribution to the N cycle in the biosphere. This symbiotic association is initiated when rhizobia infect and penetrate the root hairs, which is followed by the growth and development of root nodules, within which the infective rhizobia are established and protected. Thus, the nodule environment allows the expression and function of the enzyme complex that catalyzes N2 fixation. However, during early infection, the rhizobia find a harsh environment while penetrating the root hairs. To cope with this nuisance, the rhizobia mount a stress response known as the stringent response. In turn, the plant regulates nodulation in response to the presence of alternative sources of combined N in the surrounding medium. Control of these processes is crucial for a successful symbiosis, and here we show how the rhizobial stringent response may modulate plant defense suppression and the networks of regulation of nodulation.

Members of the Rhizobiales are polarly growing bacteria that lack homologs of the canonical Rod complex. To investigate the mechanisms underlying polar cell wall synthesis, we systematically probed the function of cell wall synthesis enzymes in the plant pathogen Agrobacterium tumefaciens. The development of fluorescent d-amino acid dipeptide (FDAAD) probes, which are incorporated into peptidoglycan by penicillin-binding proteins in A. tumefaciens, enabled us to monitor changes in growth patterns in the mutants. Use of these fluorescent cell wall probes and peptidoglycan compositional analysis demonstrate that a single class A penicillin-binding protein is essential for polar peptidoglycan synthesis. Furthermore, we find evidence of an additional mode of cell wall synthesis that requires ld-transpeptidase activity. Genetic analysis and cell wall targeting antibiotics reveal that the mechanism of unipolar growth is conserved in Sinorhizobium and Brucella. This work provides insights into unipolar peptidoglycan biosynthesis employed by the Rhizobiales during cell elongation. IMPORTANCE While the structure and function of the bacterial cell wall are well conserved, the mechanisms responsible for cell wall biosynthesis during elongation are variable. It is increasingly clear that rod-shaped bacteria use a diverse array of growth strategies with distinct spatial zones of cell wall biosynthesis, including lateral elongation, unipolar growth, bipolar elongation, and medial elongation. Yet the vast majority of our understanding regarding bacterial elongation is derived from model organisms exhibiting lateral elongation. Here, we explore the role of penicillin-binding proteins in unipolar elongation of Agrobacterium tumefaciens and related bacteria within the Rhizobiales. Our findings suggest that penicillin-binding protein 1a, along with a subset of ld-transpeptidases, drives unipolar growth. Thus, these enzymes may serve as attractive targets for biocontrol of pathogenic Rhizobiales.

When subjected to nutritional stress, bacteria modify their amino acid metabolism and cell division activities by means of the stringent response, which is controlled by the Rsh protein in alphaproteobacteria. An important group of alphaproteobacteria are the rhizobia, which fix atmospheric N(2) in symbiosis with legume plants. Although nutritional stress is common for rhizobia while infecting legume roots, the stringent response has scarcely been studied in this group of soil bacteria. In this report, we obtained a mutant with a kanamycin resistance insertion in the rsh gene of Bradyrhizobium diazoefficiens, the N(2)-fixing symbiont of soybean. This mutant was defective for type 3 secretion system induction, plant defense suppression at early root infection, and nodulation competition. Furthermore, the mutant produced smaller nodules, although with normal morphology, which led to lower plant biomass production. Soybean (Glycine max) genes GmRIC1 and GmRIC2, involved in autoregulation of nodulation, were upregulated in plants inoculated with the mutant under the N-free condition. In addition, when plants were inoculated in the presence of 10 mM NH(4)NO(3), the mutant produced nodules containing bacteroids, and GmRIC1 and GmRIC2 were downregulated. The rsh mutant released more auxin to the culture supernatant than the wild type, which might in part explain its symbiotic behavior in the presence of combined N. These results indicate that the B. diazoefficiens stringent response integrates into the plant defense suppression and regulation of nodulation circuits in soybean, perhaps mediated by the type 3 secretion system. IMPORTANCE The symbiotic N(2) fixation carried out between prokaryotic rhizobia and legume plants performs a substantial contribution to the N cycle in the biosphere. This symbiotic association is initiated when rhizobia infect and penetrate the root hairs, which is followed by the growth and development of root nodules, within which the infective rhizobia are established and protected. Thus, the nodule environment allows the expression and function of the enzyme complex that catalyzes N(2) fixation. However, during early infection, the rhizobia find a harsh environment while penetrating the root hairs. To cope with this nuisance, the rhizobia mount a stress response known as the stringent response. In turn, the plant regulates nodulation in response to the presence of alternative sources of combined N in the surrounding medium. Control of these processes is crucial for a successful symbiosis, and here we show how the rhizobial stringent response may modulate plant defense suppression and the networks of regulation of nodulation.

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger which has been associated with a motile to sessile lifestyle switch in many bacteria. Here, we review recent insights into c-di-GMP regulated processes related to environmental adaptations in alphaproteobacterial rhizobia, which are diazotrophic bacteria capable of fixing nitrogen in symbiosis with their leguminous host plants. The review centers onSinorhizobium meliloti, which in the recent years was intensively studied for its c-di-GMP regulatory network.

Bacterial cell proliferation involves cell growth and septum formation followed by cell division. For cell growth, bacteria have evolved different complex mechanisms. The most prevalent growth mode of rod-shaped bacteria is cell elongation by incorporating new peptidoglycans in a dispersed manner along the sidewall. A small share of rod-shaped bacteria, including the alphaproteobacterial Rhizobiales, grow unipolarly. Here, we identified and initially characterized a set of Rgs (rhizobial growth and septation) proteins, which are involved in cell division and unipolar growth of Sinorhizobium meliloti and highly conserved in Rhizobiales. Our data expand the knowledge of components of the polarly localized machinery driving cell wall growth and suggest a complex of Rgs proteins with components of the divisome, differing in composition between the polar cell elongation zone and the septum., Sinorhizobium meliloti is an alphaproteobacterium belonging to the Rhizobiales. Bacteria from this order elongate their cell wall at the new cell pole, generated by cell division. Screening for protein interaction partners of the previously characterized polar growth factors RgsP and RgsM, we identified the inner membrane components of the Tol-Pal system (TolQ and TolR) and novel Rgs (rhizobial growth and septation) proteins with unknown functions. TolQ, Pal, and all Rgs proteins, except for RgsE, were indispensable for S. meliloti cell growth. Six of the Rgs proteins, TolQ, and Pal localized to the growing cell pole in the cell elongation phase and to the septum in predivisional cells, and three Rgs proteins localized to the growing cell pole only. The putative FtsN-like protein RgsS contains a conserved SPOR domain and is indispensable at the early stages of cell division. The components of the Tol-Pal system were required at the late stages of cell division. RgsE, a homolog of the Agrobacterium tumefaciens growth pole ring protein GPR, has an important role in maintaining the normal growth rate and rod cell shape. RgsD is a periplasmic protein with the ability to bind peptidoglycan. Analysis of the phylogenetic distribution of the Rgs proteins showed that they are conserved in Rhizobiales and mostly absent from other alphaproteobacterial orders, suggesting a conserved role of these proteins in polar growth.

Members of the Rhizobiales (class of α-proteobacteria) display zonal peptidoglycan cell wall growth at one cell pole, contrasting with the dispersed mode of cell wall growth along the sidewalls of many other rod-shaped bacteria. Here we show that the seven-transmembrane receptor (7TMR) protein RgsP (SMc00074), together with the putative membrane-anchored peptidoglycan metallopeptidase RgsM (SMc02432), have key roles in unipolar peptidoglycan formation during growth and at mid-cell during cell division in Sinorhizobium meliloti. RgsP is composed of a periplasmic globular 7TMR-DISMED2 domain, a membrane-spanning region, and cytoplasmic PAS, GGDEF and EAL domains. The EAL domain confers phosphodiesterase activity towards the second messenger cyclic di-GMP, a key regulatory player in the transition between bacterial lifestyles. RgsP and RgsM localize to sites of zonal cell wall synthesis at the new cell pole and cell divison site, suggesting a role in cell wall biogenesis. The two proteins are essential for cell wall biogenesis and cell growth. Cells depleted of RgsP or RgsM had an altered muropeptide composition and RgsM binds to peptidoglycan. RgsP and RgsM orthologs are functional when interchanged between α-rhizobial species pointing to a conserved mechanism for cell wall biogenesis/remodeling within the Rhizobiales. Overall, our findings suggest that RgsP and RgsM contribute to the regulation of unipolar cell wall biogenesis in α-rhizobia., Author summary Bacteria face the challenge of maintaining their peptidoglycan cell wall integrity during growth and division. The enzymes involved in cell wall biogenesis are tightly regulated and targeting of these enzymes by β-lactams cause cell death. In contrast to the well-characterized mode of dispersed cell wall formation in many rod-shaped bacteria, the mechanisms controlling polar cell wall formation in α-proteobacteria are largely unknown. Seven-transmembrane receptors (7TMRs) are widespread in eukaryotes and prokaryotes but only few of them have been functionally studied in bacteria. Here we suggest that a 7TMR-DISM protein cooperates with a putative peptidoglycan-hydrolyzing protein, to facilitate unipolar cell wall formation and cell division in the Rhizobiales. This 7TMR-DISM protein also contributes to degradation of the second messenger cyclic di-GMP.

Sinorhizobium fredii HH103 is a fast-growing rhizobial strain infecting a broad range of legumes including both American and Asiatic soybeans. In this work, we present the sequencing and annotation of the HH103 genome (7.25 Mb), consisting of one chromosome and six plasmids and representing the structurally most complex sinorhizobial genome sequenced so far. Comparative genomic analyses of S. fredii HH103 with strains USDA257 and NGR234 showed that the core genome of these three strains contains 4,212 genes (61.7% of the HH103 genes). Synteny plot analysis revealed that the much larger chromosome of USDA257 (6.48 Mb) is colinear to the HH103 (4.3 Mb) and NGR324 chromosomes (3.9 Mb). An additional region of the USDA257 chromosome of about 2 Mb displays similarity to plasmid pSfHH103e. Remarkable differences exist between HH103 and NGR234 concerning nod genes, flavonoid effect on surface polysaccharide production, and quorum-sensing systems. Furthermore a number of protein secretion systems have been found. Two genes coding for putative type III–secreted effectors not previously described in S. fredii, nopI and gunA, have been located on the HH103 genome. These differences could be important to understand the different symbiotic behavior of S. fredii strains HH103, USDA257, and NGR234 with soybean.

Cyclic dimeric GMP (c-di-GMP) has emerged as a key regulatory player in the transition between planktonic and sedentary biofilm-associated bacterial lifestyles. It controls a multitude of processes including production of extracellular polysaccharides (EPSs). The PilZ domain, consisting of an N-terminal "RxxxR" motif and a β-barrel domain, represents a prototype c-di-GMP receptor. We identified a class of c-di-GMP-responsive proteins, represented by the AraC-like transcription factor CuxR in plant symbiotic α-proteobacteria. In Sinorhizobium meliloti, CuxR stimulates transcription of an EPS biosynthesis gene cluster at elevated c-di-GMP levels. CuxR consists of a Cupin domain, a helical hairpin, and bipartite helix-turn-helix motif. Although unrelated in sequence, the mode of c-di-GMP binding to CuxR is highly reminiscent to that of PilZ domains. c-di-GMP interacts with a conserved N-terminal RxxxR motif and the Cupin domain, thereby promoting CuxR dimerization and DNA binding. We unravel structure and mechanism of a previously unrecognized c-di-GMP-responsive transcription factor and provide insights into the molecular evolution of c-di-GMP binding to proteins.

Quorum sensing (QS) using N-acyl homoserine lactones (AHLs) as signal molecules is a common strategy used by diverse Gram-negative bacteria. A widespread mechanism of AHL sensing involves binding of these molecules by cytosolic LuxR-type transcriptional regulators, which requires uptake of external AHLs. The outer membrane is supposed to be an efficient barrier for diffusion of long-chain AHLs. Here we report evidence that in Sinorhizobium meliloti, sensing of AHLs with acyl chains composed of 14 or more carbons is facilitated by the outer membrane protein FadLSm, a homolog of the Escherichia coli FadLEc long-chain fatty acid transporter. The effect of fadLSm on AHL sensing was more prominent for longer and more hydrophobic signal molecules. Using reporter gene fusions to QS target genes, we found that fadLSm increased AHL sensitivity and accelerated the course of QS. In contrast to FadLEc, FadLSm did not support uptake of oleic acid, but did contribute to growth on palmitoleic acid. FadLSm homologs from related symbiotic α-rhizobia and the plant pathogen Agrobacterium tumefaciens differed in their ability to facilitate long-chain AHL sensing or to support growth on oleic acid. FadLAt was found to be ineffective toward long-chain AHLs. We obtained evidence that the predicted extracellular loop 5 of FadLSm and further α-rhizobial FadL proteins contains determinants of specificity to long-chain AHLs. Replacement of a part of loop 5 by the corresponding region from α-rhizobial FadL proteins transferred sensitivity for long-chain AHLs to FadLAt.

To identify physiological processes affected by cAMP in the plant-symbiotic nitrogen-fixing α-proteobacterium Sinorhizobium meliloti Rm2011, cAMP levels were artificially increased by overexpression of its cognate adenylate/guanylate cyclase gene cyaJ. This resulted in high accumulation of cAMP in the culture supernatant, decreased swimming motility and increased production of succinoglycan, an exopolysaccharide involved in host invasion. Weaker, similar phenotypic changes were induced by overexpression of cyaB and cyaG1. Effects on swimming motility and succinoglycan production were partially dependent on clr encoding a cyclic AMP receptor-like protein. Transcriptome profiling of an cyaJ-overexpressing strain identified 72 upregulated and 82 downregulated genes. A considerable number of upregulated genes are related to polysaccharide biosynthesis and osmotic stress response. These included succinoglycan biosynthesis genes, genes of the putative polysaccharide synthesis nodP2-exoF3 cluster and feuN, the first gene of the operon encoding the FeuNPQ regulatory system. Downregulated genes were mostly related to respiration, central metabolism and swimming motility. Promoter-probe studies in the presence of externally added cAMP revealed 18 novel Clr-cAMP-regulated genes. Moreover, the addition of cGMP into the growth medium also promoted clr-dependent gene regulation. In vitro binding of Clr-cAMP and Clr-cGMP to the promoter regions of SMc02178, SMb20906,SMc04190, SMc00925, SMc01136 and cyaF2 required the DNA motif (A/C/T)GT(T/C)(T/C/A)C (N4) G(G/A)(T/A)ACA. Furthermore, SMc02178, SMb20906,SMc04190and SMc00653 promoters were activated by Clr-cAMP/cGMP in Escherichia coli as heterologous host. These findings suggest direct activation of these 7 genes by Clr-cAMP/cGMP.

Sinorhizobium meliloti is a symbiotic soil bacterium that forms nitrogen-fixing nodules on roots of leguminous plants, including Medicago truncatula (barrel medic), and M. sativa (alfalfa). The Sinorhizobium-Medicago symbiosis is an important symbiosis model system. Knowledge gained from this system can be extended to other agriculturally important "rhizobial" symbioses. Since the publication of the S. meliloti genome in 2001, many new genetic, biochemical and physiological data have been generated. Effective methods to organize, store, and mine this postgenome data are crucial for continued success of the S. meliloti model system. In 2009, we introduced a portal for rhizobial genomes, RhizoGATE (Becker et al., J. Biotechnol. 140, 45-50). The RhizoGATE portal combines continuously updated S. meliloti genome annotation with postgenome data resources. Here we report integration of a new component, RhizoRegNet, to RhizoGATE. RhizoRegNet combines transcriptome data and operon predictions with published data on regulatory interactions. By allowing searching and visualisation of complex transcriptional regulatory networks, RhizoRegNet advances our understanding of transcriptional regulation in S. meliloti. The current version of RhizoRegNet is divided into 13 functional modules containing information for 114 regulators, 475 regulated genes, and 178 transcription factor binding motifs. In this report, we provide an example of how RhizoRegNet facilitates visualisation and analysis of the regulatory network for exopolysaccharide biosynthesis and motility. Presently, RhizoRegNet contains regulatory network information for S. meliloti and the closely related bacterium, S. medicae, but can be expanded to include other rhizobial species. (C) 2010 Elsevier B. V. All rights reserved.

Sinorhizobium meliloti Rm2011 responds to sudden shifts to nitrogen or carbon starvation conditions by an accumulation of the stringent response alarmone ppGpp and remodelling of the transcriptome. The gene product of relA, Rel(Sm) , responsible for synthesis of ppGpp, shows functional similarities to E. coli SpoT. Using promoter-egfp gene fusions, we showed that in Rm2011 relA is expressed at a low rate, as a readthrough from the rpoZ promoter and from its own weak promoter. The low level of relA expression is physiologically relevant, since overexpression of Rel(Sm) inhibits ppGpp accumulation. The N-terminal portion of Rel(Sm) is required for ppGpp degradation in nutrient-sufficient cells and might be involved in regulation of the ppGpp synthase and hydrolase activities of the protein. Expression profiling of S. meliloti subjected to sudden nitrogen or carbon downshifts revealed that repression of 'house-keeping' genes is largely dependent on relA whereas activation of gene targets of the stress sigma factor RpoE2 occurred independently of relA. The regulatory genes nifA, ntrB, aniA and sinR, as well as genes related to modulation of protein biosynthesis and nucleotide catabolism, were induced in a relA-dependent manner. dksA was required for the majority of the relA-dependent regulations.

In order to enter symbiosis with its legume partner, Sinorhizobium meliloti requires regulatory systems for the appropriate responses to its environment. For example, motility is required for the chemotactic movement of bacteria toward the compounds released by its host, and exopolysaccharides (EPS) are required for bacterial attachment to the root or for invasion of the infection thread. Previous research has shown that ExoR/ExoS/ChvI as well as the ExpR/Sin quorum-sensing system inversely regulate both motility and EPS production, although the regulation mechanisms were unknown. We were able to attribute the ExpR-mediated regulation of motility to the ability of ExpR to bind a DNA sequence upstream of visN when activated by N-acyl-homoserine lactone. Furthermore, MucR, previously characterized as a regulator of EPS production, also affected motility. MucR inhibited expression of rem encoding an activator of motility gene expression and, consequently, the expression of Rem-regulated genes such as flaF and flgG. Binding of MucR to the rem promoter region was demonstrated and a sequence motif similar to the previously identified MucR binding consensus was identified within this region. The swarming ability of S. meliloti Rm2011 was shown to depend on a functional ExpR/Sin quorum-sensing system and the production of both flagella and EPS. Finally, we propose a model for the coordination of motility and EPS synthesis in S. meliloti.

The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti possesses the Sin quorum-sensing system based on N -acyl homoserine lactones (AHLs) as signal molecules. The Sin system consists of SinI, the AHL synthase, and SinR, the LuxR-type regulator. This system regulates the expression of a multitude of S. meliloti genes through ExpR, another LuxR-type regulator. Analysis of the activity of the sinI promoter showed that the expression of sinI is dependent on sinR and enhanced by a combination of expR and Sin AHLs. The characterization of the ExpR binding site upstream of sinI and the identification of binding sites upstream of the galactoglucan biosynthesis genes wgaA ( expA1 ) and wgeA ( expE1 ) allowed the definition of a consensus sequence for these binding sites. Based on this consensus, two additional ExpR binding sites in the promoter regions of exoI and exsH , two genes related to the production of succinoglycan, were found. The specific binding of ExpR to the wgaA and wgeA promoters was enhanced in the presence of oxo-C 14 -HL. Positive regulation of the galactoglucan biosynthesis genes by ExpR was shown to be dependent on WggR (ExpG) and influenced by MucR, both of which are previously characterized regulators of these genes. Based on these results, a reworked model of the Sin-ExpR quorum-sensing regulation scheme of galactoglucan production in S. meliloti is suggested.

Sinorhizobium meliloti undergoes major lifestyle changes between planktonic states, biofilm formation, and symbiosis with leguminous plant hosts. In many bacteria, the second messenger 3′,5′-cyclic di-GMP (c-di-GMP, or cdG) promotes a sessile lifestyle by regulating a plethora of processes involved in biofilm formation, including motility and biosynthesis of exopolysaccharides (EPS). Here, we systematically investigated the role of cdG in S. meliloti Rm2011 encoding 22 proteins putatively associated with cdG synthesis, degradation, or binding. Single mutations in 21 of these genes did not cause evident changes in biofilm formation, motility, or EPS biosynthesis. In contrast, manipulation of cdG levels by overproducing endogenous or heterologous diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) affected these processes and accumulation of N -Acyl-homoserine lactones in the culture supernatant. Specifically, individual overexpression of the S. meliloti genes pleD , SMb20523 , SMb20447 , SMc01464 , and SMc03178 encoding putative DGCs and of SMb21517 encoding a single-domain PDE protein had an impact and resulted in increased levels of cdG. Compared to the wild type, an S. meliloti strain that did not produce detectable levels of cdG (cdG 0 ) was more sensitive to acid stress. However, it was symbiotically potent, unaffected in motility, and only slightly reduced in biofilm formation. The SMc01790-SMc01796 locus, homologous to the Agrobacterium tumefaciens uppABCDEF cluster governing biosynthesis of a unipolarly localized polysaccharide, was found to be required for cdG-stimulated biofilm formation, while the single-domain PilZ protein McrA was identified as a cdG receptor protein involved in regulation of motility. IMPORTANCE We present the first systematic genome-wide investigation of the role of 3′,5′-cyclic di-GMP (c-di-GMP, or cdG) in regulation of motility, biosynthesis of exopolysaccharides, biofilm formation, quorum sensing, and symbiosis in a symbiotic alpha-rhizobial species. Phenotypes of an S. meliloti strain unable to produce cdG (cdG 0 ) demonstrated that this second messenger is not essential for root nodule symbiosis but may contribute to acid tolerance. Our data further suggest that enhanced levels of cdG promote sessility of S. meliloti and uncovered a single-domain PilZ protein as regulator of motility.

The global response to phosphate starvation was analysed at the transcriptional level in two closely related strains of Sinorhizobium meliloti, Rm1021 and Rm2011. The Pho regulon is known to be induced by PhoB under conditions of phosphate limitation. Ninety-eight genes were found to be significantly induced (more than three-fold) in a phoB -dependent manner in phosphate-stressed cells, and phoB -independent repression of 86 genes was observed. Possible roles of these genes in the phosphate stress response are discussed. Twenty new putative PHO box sequences were identified in regions upstream of 17 of the transcriptional units that showed phoB -dependent, or partially phoB -dependent, regulation, indicating direct regulation of these genes by PhoB. Despite the overall similarity between the phosphate stress responses in Rm1021 and Rm2011, lower induction rates were found for a set of phoB -dependent genes in Rm1021. Moreover, Rm1021 exhibited moderate constitutive activation of 12 phosphate starvation-inducible, phoB -dependent genes when cells were grown in a complex medium. A 1-bp deletion was observed in the pstC ORF in Rm1021, which results in truncation of the protein product. This mutation is probably responsible for the expression of phosphate starvation-inducible genes in Rm1021 in the absence of phosphate stress.

Sinorhizobium meliloti is an α-proteobacterium that alternates between a free-living phase in bulk soil or in the rhizosphere of plants and a symbiotic phase within the host plant cells, where the bacteria ultimately differentiate into nitrogen-fixing organelle-like cells, called bacteroids. As a step toward understanding the physiology of S. meliloti in its free-living and symbiotic forms and the transition between the two, gene expression profiles were determined under two sets of biological conditions: growth under oxic versus microoxic conditions, and in free-living versus symbiotic state. Data acquisition was based on both macro- and microarrays. Transcriptome profiles highlighted a profound modification of gene expression during bacteroid differentiation, with 16% of genes being altered. The data are consistent with an overall slow down of bacteroid metabolism during adaptation to symbiotic life and acquisition of nitrogen fixation capability. A large number of genes of unknown function, including potential regulators, that may play a role in symbiosis were identified. Transcriptome profiling in response to oxygen limitation indicated that up to 5% of the genes were oxygen regulated. However, the microoxic and bacteroid transcriptomes only partially overlap, implying that oxygen contributes to a limited extent to the control of symbiotic gene expression.

Based on the complete Sinorhizobium meliloti genome sequence we established DNA microarrays as a comprehensive tool for systematic genome-wide gene expression analysis in S. meliloti 1021. For these PCR fragment-based microarrays, called Sm6kPCR, a collection of probes for the 6207 predicted protein-coding genes consisting of 6046 gene-specific PCR fragments and 161 70 mer oligonucleotides was arrayed in high density on glass slides. To obtain these PCR fragments primer pairs were designed to amplify internal gene-specific DNA fragments of 80–350 bp. Additionally, these primers were characterized by a 5′ extension that allowed for reamplification using standard primers after the first amplification employing the specific primers. In order to ascertain the quality of the Sm6kPCR microarrays and to validate gene expression studies in S. meliloti parallel hybridizations based on RNA samples obtained from cells cultured under identical conditions were performed. In addition, gene expression in S. meliloti in response to an osmotic upshift imposed by the addition of 0.38 M NaCl was monitored. 137 genes were identified showing significant changes in gene expression resulting from the osmotic upshift. From these genes 52 were induced and 85 genes were repressed. Among the genes displaying different RNA levels some functional groups could be identified that are particularly remarkable. Repression was observed for 8 genes related to motility and chemotaxis, 7 genes encoding amino acid biosynthesis enzymes and 15 genes involved in iron uptake whereas 14 genes involved in transport of small molecules and 4 genes related to polysaccharide biosynthesis were induced.

Background Sinorhizobium meliloti is a soil-dwelling α-proteobacterium that possesses a large, tripartite genome and engages in a nitrogen fixing symbiosis with its plant hosts. Although much is known about this important model organism, global characterization of genetic regulatory circuits has been hampered by a lack of information about transcription and promoters. Results Using an RNAseq approach and RNA populations representing 16 different growth and stress conditions, we comprehensively mapped S. meliloti transcription start sites (TSS). Our work identified 17,001 TSS that we grouped into six categories based on the genomic context of their transcripts: mRNA (4,430 TSS assigned to 2,657 protein-coding genes), leaderless mRNAs (171), putative mRNAs (425), internal sense transcripts (7,650), antisense RNA (3,720), and trans-encoded sRNAs (605). We used this TSS information to identify transcription factor binding sites and putative promoter sequences recognized by seven of the 15 known S. meliloti σ factors σ70, σ54, σH1, σH2, σE1, σE2, and σE9). Altogether, we predicted 2,770 new promoter sequences, including 1,302 located upstream of protein coding genes and 722 located upstream of antisense RNA or trans-encoded sRNA genes. To validate promoter predictions for targets of the general stress response σ factor, RpoE2 (σE2), we identified rpoE2-dependent genes using microarrays and confirmed TSS for a subset of these by 5′ RACE mapping. Conclusions By identifying TSS and promoters on a global scale, our work provides a firm foundation for the continued study of S. meliloti gene expression with relation to gene organization, σ factors and other transcription factors, and regulatory RNAs.

Sinorhizobium meliloti Rm2011 responds to sudden shifts to nitrogen or carbon starvation conditions by an accumulation of the stringent response alarmone ppGpp and remodelling of the transcriptome. The gene product of relA, Rel(Sm) , responsible for synthesis of ppGpp, shows functional similarities to E. coli SpoT. Using promoter-egfp gene fusions, we showed that in Rm2011 relA is expressed at a low rate, as a readthrough from the rpoZ promoter and from its own weak promoter. The low level of relA expression is physiologically relevant, since overexpression of Rel(Sm) inhibits ppGpp accumulation. The N-terminal portion of Rel(Sm) is required for ppGpp degradation in nutrient-sufficient cells and might be involved in regulation of the ppGpp synthase and hydrolase activities of the protein. Expression profiling of S. meliloti subjected to sudden nitrogen or carbon downshifts revealed that repression of 'house-keeping' genes is largely dependent on relA whereas activation of gene targets of the stress sigma factor RpoE2 occurred independently of relA. The regulatory genes nifA, ntrB, aniA and sinR, as well as genes related to modulation of protein biosynthesis and nucleotide catabolism, were induced in a relA-dependent manner. dksA was required for the majority of the relA-dependent regulations.

Sinorhizobium meliloti is a symbiotic soil bacterium of the alphaproteobacterial subdivision. Like other rhizobia, S. meliloti induces nitrogen-fixing root nodules on leguminous plants. This is an ecologically and economically important interaction, because plants engaged in symbiosis with rhizobia can grow without exogenous nitrogen fertilizers. The S. meliloti–Medicago truncatula (barrel medic) association is an important symbiosis model. The S. meliloti genome was published in 2001, and the M. truncatula genome currently is being sequenced. Many new resources and data have been made available since the original S. meliloti genome annotation and an update was needed. In June 2008, we submitted our annotation update to the EMBL and NCBI databases. Here we describe this new annotation and a new webbased portal RhizoGATE. About 1000 annotation updates were made; these included assigning functions to 313 putative proteins, assigning EC numbers to 431 proteins, and identifying 86 new putative genes. RhizoGATE incorporates the new annotion with the S. meliloti GenDB project, a platform that allows annotation updates in real time. Locations of transposon insertions, plasmid integrations, and array probe sequences are available in the GenDB project. RhizoGATE employs the EMMA platform for management and analysis of transcriptome data and the IGetDB data warehouse to integrate a variety of heterogeneous external data sources. © 2008 Elsevier B.V. All rights reserved.

In different bacterial species, ccmIEFH genes have been suggested to code for subunits of a bacterial haem-lyase catalyzing the covalent attachment of haem to c-type apoproteins. In Rhizobium etli CE3 there are two copies of ccmIEFH: one in the chromosome and the other located in plasmid pf. However, the null phenotype of chromosomal ccmF mutant indicates that the gene locus of plasmid pf is not functional. Two ccmI chromosomal mutants, previously isolated, produced detectable levels of c-type cytochromes under certain culture conditions in contrast with the ccmF mutant, suggesting that ccmF could be transcribed independently. The transcriptional organization of ccmIEFH operon was established. Two promoters from the chromosomal locus were mapped by primer extension, one located upstream of ccmI and the second located upstream of ccmF. The regulation of the expression of both promoters was studied using appropriate lacZ gene fusions (ccmI–lacZ and ccmEF–lacZ). The ccmI–lacZ gene fusion was expressed in complex medium, during exponential growth, under microaerobic conditions and in a R. etli mutant that accumulates reducing power, conditions where a higher respiration rate could be limited by c-type cytochrome content. The ccmEF–lacZ fusion was also primarily expressed in complex medium and under microaerophilic conditions. The finding of two independent promoters in this gene locus could suggest that the step catalyzed by CcmFH could be a rate-limiting step for c-type cytochrome assembly under certain culture conditions.